PROJECT SUMMARY This application is being submitted in response to NOT-OD-19-071. Down syndrome (DS) is the most commonly occurring chromosomal abnormality in live births in the US and worldwide. Up to 50% of DS patients suffer from congenital heart disease (CHD) such as ventricular septal defect, atrioventricular septal defect, and tetralogy of Fallot. Complications from these cardiac anomalies cause major morbidities and mortalities in this population. Yet, it is still unknown why the presence of extra chromosome 21 leads to the development of these potentially fatal CHD, due in part to a lack of adequate in vivo model overcoming interspecies differences in genetic composition and inability to access DS-affected human cardiomyocytes. In this Supplement to the Parent R01 HL141371, we propose to leverage patient-derived human induced pluripotent stem cell (iPSC) platform towards studying mechanisms of CHD in DS patients. We hypothesize overexpression of cardiac-specific, dosage-sensitive trisomic genes on chr21 leads to heart defects through impaired cardiac crosstalk and myocyte maturation. Aim 1 will generate a biorepository of 40 DS-specific iPSC lines. To investigate the role of intercellular crosstalk in the pathogenesis of DS-related CHD, we will engineer iPSC-cardiac organoids resembling the heart tissue composition of cardiomyocytes, endothelial cells, and fibroblasts and determine molecular and functional phenotypes of cardiac organoids derived from the DS iPSCs. In Aim 2, we will investigate the mechanism of DS-related CHD using a pan-omic approach. The mechanisms of identified gene candidates will be further investigated through genome editing strategy. Completing the aims of this supplement will likely increase our understanding of DS-related CHD as well as broaden the overall impact of the parent R01 award. In the parent award, we are using iPSC technology to identify mechanisms of genetic cardiomyopathy in vitro and dissecting the role of crosstalk between cardiovascular cell types in pathogenesis. Mechanism underlying DS-related CHD involves complex inter- cellular communication leading to developmental and structural anomalies. Hence, we are confident that a comparative in vitro and bioinformatics analysis utilizing both DS and non-DS CHD iPSC-derived cardiomyocytes will likely extend our understanding of CHD as well as facilitate the discovery of novel genes and pathways that may be critical in its pathogenesis. In summary, this Administrative Supplement proposal will create novel opportunities to build a massive functional and sequencing data as well as iPSC repository that will be broadly shared with investigators of CHD and DS. The combination of deep clinical phenotyping, the use of iPSC-based engineered cardiac organoid tissues, multi-omic profiling, and cross-talk signaling studies will undoubtedly help to move this field forward and better understand DS-related cardiac pathologies.